Amino acid, derivatives, processes for their preparation and their therapeutic application

ABSTRACT

New amino acid derivatives, processes for their preparation and their therapeutic application. 
     Amino acid derivatives corresponding to the general formulae ##STR1## These derivatives may be used as medicaments which exhibit an enkephalinase-inhibitory activity.

The present invention relates to new amino acid derivatives, toprocesses for their preparation and to their therapeutic application.

The new derivatives according to the invention are inhibitors ofenkephalinase, which is an enzyme which degrades enkephalins.

Methionine-enkephalin and leucine-enkephalin are pentapeptides whichhave been discovered in the brain and which are endogenous ligands ofthe morphinic receptor (J. Hughes et al., Nature 258, 577-579 (1975)).

These peptides are considered as neuromediators having inhibitory actionon the release of other neuro-transmitters (J. Hughes, Nature 278, 394(1979); S. H. Snyder, Nature 278, 13 (1979)). It has been demonstratedthat enkephalins are rapidly degraded by a carboxydipeptidase("enkephalinase") which releases Tyr-Gly-Gly and Phe-Met residues (B.Malfroy et al., Nature, 276, p. 523-526 (1978)).

Compounds which are capable of inhibiting enkephalinase may thus prolongthe effects of the endogenous enkephalins or may potentiate the actionof synthetic analogues administered in an exogenous fashion. Thesecompounds are thus capable of replacing morphinic agents in all theirproperties without having any of the disadvantages thereof, inparticular regarding the phenomena of addiction and dependency.

The development of compounds capable of inhibiting the degradation ofthe enkephalins by the enzyme enkephalinase has been the subject ofextensive research.

By way of illustration of the state of the art, there may be mentionedEuropean Patent Applications 0,038,758 and 0,082,088 (Roques et al.)which describe amino acid derivatives and, more particularly,mercaptoalkanoyl and acylmercaptoalkanoyl amino acid derivatives havingan enkephalinase-inhibitory activity.

U.S. Pat. No. 4,401,677 (Greenberg et al.) describes variousmercaptoalkanoyl amino acids which are useful as analgesic agents, onaccount of their enkephalinase-inhibitory activity.

There may also be mentioned European Patent Application No. 0,136,883(Delaney et al.) which describes enkephalinase inhibitors correspondingto the general formula ##STR2## in which R₁ represents a hydrogen atomor an acyl group, R₂ represents, inter alia, a benzyl radical, thebenzene ring being optionally mono- or polysubstituted with a halogenatom, a trifluoromethyl group, a nitro group, a lower alkyl group or alower alkoxy group, R₃ represents, inter alia, a hydrogen atom or alower alkyl, R₄ represents a hydroxyl, esters or amides and n is aninteger between 1 and 15.

The new enkephalinase inhibitors in accordance with the invention havethe advantage, with respect to the enkephalinase inhibitors knownpreviously, such as those described in the abovementioned patents orpatent applications, of lesser bonding to plasma proteins and thus ofgreater activity, by virtue in particular of the presence in theirstructure of an ethylenic double bond at the carbon alpha to thecarbonyl group of the amide function.

The new amino acid derivatives in accordance with the inventioncorrespond to the general formulae ##STR3## in which R₁ represents ahydrogen atom; a phenyl group which is optionally mono- orpolysubstituted with a halogen atom, a trifluoromethyl group, a nitrogroup, a cyano group or an amino group, a lower alkyl group or a lowerphenylalkylene group; the group ##STR4## where R'₁ represents a hydrogenatom, a lower alkyl group, a phenyl group or a lower phenylalkylenegroup; a group ##STR5## where A, B and n₃ have the meanings given below

    ______________________________________                                        A               B      n.sub.3                                                ______________________________________                                        O               O      1                                                      O               CH.sub.2                                                                             1                                                      CH.sub.2        CH.sub.2                                                                             1                                                      O               O      2                                                      CH.sub.2        CH.sub.2                                                                             2                                                      O               CH.sub.2                                                                             2                                                      ______________________________________                                    

a biphenyl group, alpha and beta naphthyl, n₁ varies from 0 to 10

n₂ varies from 1 to 10

R₂ represents a hydrogen atom; a lower alkyl group; a lowerhydroxyalkylene group; a phenyl group; a lower phenylalkylene group; alower hydroxyphenylalkylene group; a lower aminoalkylene group; a lowerguanidinoalkylene group; a lower mercaptoalkylene group; a lowerthioalkylene lower alkyl group; a lower imidazolylalkylene group; alower indolylalkylene group; a lower carbamylalkylene group; a lowercarboxyalkylene group;

R₃ also represents a hydrogen atom or one of the groups mentioned abovefor the definition of R₂ ;

R₄ represents a hydrogen atom, a linear or branched aliphatic acylradical, an aromatic acyl radical which is optionally mono- orpolysubstituted, or a linear or branched acyl radical containing one ormore oxygen atoms;

R₅ represents a hydrogen atom; a linear or branched lower alkyl group; aphenyl group or a lower phenylalkylene group, the two last-mentionedgroups being optionally mono- or polysubstituted on the phenyl ring; alinear or branched substituent containing one or more oxygen atoms.

Lower alkyl groups is understood to refer to alkyl groups having alinear or branched chain containing 1 to 6 carbon atoms and preferably 1to 4 carbon atoms.

Lower alkylene groups is understood to refer to alkylene groupscontaining 1 to 6 carbon atoms and preferably 1 to 4 carbon atoms.

Fluorine is particularly preferred as halogen atom.

Acetyl, propionyl and pivaloyl groups may be mentioned as aliphatic acylgroup, the acetyl group being preferred. The benzoyl group may bementioned as aromatic acyl group.

Among the compounds of formulae (Ia) and (Ib) defined above, onepreferred class of compounds comprises the derivatives in which:

R₁ represents a phenyl group which is optionally mono- orpolysubstituted with a halogen atom or with the trifluoromethyl group,the group ##STR6## where R'₁ represents a lower alkyl group, a phenylgroup; the group ##STR7## where A and B represent oxygen and n₃ is equalto 1 or 2; a biphenyl group, alpha and beta naphthyl;

R₂ and R₃ represent a hydrogen atom or a lower alkyl radical;

R₄ represents a hydrogen atom, a linear or branched aliphatic acylradical or an aromatic acyl radical;

R₅ represents a hydrogen atom, a lower alkyl radical or a phenylradical;

n₁ is equal to 0 or 1;

n₂ is equal to 1 or 2.

Among the compounds of formulae (Ia) and (Ib), those which describe thefollowing amino acid structures: beta-alanine and gamma-aminobutyricacid, are preferred.

Among the more particularly preferred compounds of formulae (Ia) and(Ib), there may be mentioned (in the formulae given below, Ph denotesthe phenyl group): ##STR8##

The compounds of formulae (Ia) and (Ib) possess zero, one or twoasymmetric centres. They thus exist in racemic mixture form or inoptically pure form. All these compounds also enter into the scope ofthe present invention.

The present invention also relates to the processes for preparing thecompounds of formulae (Ia) and (Ib).

The process for preparing the compounds of formula (Ib) (compounds whichpossess a double bond of (Z) configuration), is characterized in that itconsists successively: a) in carrying out an allylic bromination of anethylenic acid of (E) configuration of formula (II) ##STR9## in which R₁has the abovementioned meaning, with a brominating agent such asN-bromosuccinimide in the presence of a catalytic amount of benzoylperoxide in order to form an acid of formula (III) ##STR10## b) insubstituting the bromine of the acid of formula (III), preferably with athioacid R₄ -SH, in order to form the thioacyl ethylenic acid of (Z)configuration of formula (IV) ##STR11## where R₄ has the abovementioneddefinition, c) in coupling the acid of formula (IV) with the desiredamino ester salt of formula (V) ##STR12## where R₂, R₃, R₅ and n₂ havethe abovementioned meanings and X represents, for example, halides (inparticular chloride), benzenesulphonate, methanesulphonate andtoluenesulphonate, in order to form the compound of (Z) configuration offormula (Ib) ##STR13## where R₄ and R₅ are not hydrogen atoms, d) insubjecting the compound of formula (Ib) to an alkaline deprotection inorder to form the compounds of (Z) configuration of formula (Ib) whereR₄ and R₅ are hydrogen atoms ##STR14##

The ethylenic acid of (E) configuration of formula (II) (step a) may beprepared, for example, by a Perkin reaction as described in Org. React.1, 210-216, 1942.

Substitution of the bromine of the acid of formula (III) with a thioacid(step b) may be carried out according to a method using potassiumcarbonate and sodium hydrogen carbonate in water or according to amethod using diisopropylethylamine in tetrahydrofuran.

The condensation reaction in c) (coupling of the acid of formula (IV)with the amino ester salt of formula (V)) is preferably carried out byusing the dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBT)process.

The alkaline deprotection in d) is preferably carried out with aqueoussodium hydroxide solution in methyl alcohol.

The process for preparing the compounds of formula (Ia) (double bond of(E) configuration) is characterized in that it consists successively: a)in isomerizing the ethylenic acid of (Z) configuration of formula (IV)##STR15## where R₄, R₁ and n₁ have the abovementioned meanings, forexample using an ultra-violet (U.V.) lamp, and then in separating the(E/Z) mixture of isomers obtained, by an amine such as cyclohexylamine,in order to obtain the thioacyl ethylenic acid of (E) configuration offormula (VI) ##STR16## b) in coupling the acid of formula (VI) with thedesired amino ester salt of formula (V), in the presence of a couplingagent such as dicyclohexylcarbodiimide, in order to obtain the compoundof formula (Ia) of (E) configuration where R₄ and R₅ are not hydrogenatoms ##STR17## c) and then in subjecting the compound of formula (Ia)to an alkaline deprotection, in order to obtain the compounds of (E)configuration of formula (Ia) where R₄ and R₅ are hydrogen atoms##STR18##

The alkaline deprotection to which the compound of formula (Ia) issubjected is preferably carried out using lithium hydroxide in mixedsolvents such as tetrahydrofuran and water.

According to one variant, the compounds of formula (Ia) are prepared: a)by isomerizing the ethylenic acid of formula (IV) possessing a (Z)configuration using an ultra-violet (U.V.) lamp in order to obtain thethioacyl ethylenic acid in the form of a mixture of isomers of (Z/E)configuration of formula (VII) ##STR19## where R₄, R₁ and n₁ have theabovementioned meanings, b) by coupling the acid of formula (VII) withthe desired amino ester salt of formula (V) in the presence of acoupling agent such as dicyclohexylcarbodiimide, in order to obtain thecompound of formula (VIII) consisting of a (Z/E) mixture of isomers##STR20## c) by separating the (Z/E) mixture of isomers of the compoundof formula (VIII), for example by flash chromatography on silica or byfractional recrystallization, in order to obtain the compound of (E)configuration of formula (Ia) where R₄ and R₅ are not hydrogen atoms##STR21##

According to another embodiment, the compounds of formula (Ia) where R₄and R₅ are not hydrogen, are prepared;

a) by isomerizing the compound of formula (Ib) possessing a double bondof (Z) configuration ##STR22## for example using an ultra-violet (U.V.)lamp, preferably in the presence of boron trifluoride etherate, in orderto obtain the compound of formula (VIII) consisting of a (Z/E) mixtureof isomers ##STR23## b) by separating the (Z/E) mixture of isomers ofthe compound of formula (VIII), for example by flash chromatography onsilica or by fractional recrystallization, in order to obtain thecompound of (E) configuration of formula (Ia) where R₄ and R₅ are nothydrogen atoms ##STR24##

Several examples of implementation of the invention will be given below,in a non-limiting manner. In the examples given below, the followingabbreviations have been used for the description of the spectra:

    ______________________________________                                        s: singlet    t: triplet   m: multiplet                                       d: doublet    q: quadruplet                                                                              app: apparent                                      ______________________________________                                    

The chemical shifts are expressed in ppm. The melting points aremeasured on a KOFLER block.

Example 1: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate

A) Preparation of (Z)-2-bromomethyl-3-propenoic acid

A mixture of 2 g (12.34 mmol) of (E)-2-methylcinnamic acid, 2.19 g(12.34 mmol) of N-bromosuccinimide (NBS) and a catalytic amount ofbenzoyl peroxide in 6 ml of CCl₄ is heated at reflux for 6 hours. Theresidue is filtered and washed with ether. The organic phase is washedsuccessively with aqueous 1N HCl solution and then with water. It isdried over MgSO₄, filtered and evaporated to dryness. Yield=57%(recrystallized in ether) m.p. 168° C. IR (nujol): 1665 cm⁻¹ ¹ H NMR(CDCl₃ /TMS): 10.0 (s, 1H); 7.9 (s, 1H); 7.8 to 7.2 (m, 5H); 4.0 (s,2H).

B) Preparation of (Z)-2-acetylthiomethyl-3-phenylpropenoic acid

2.1 g of the acid obtained above (8.70 mmol), 0.73 g (8.70 mmol) ofNaHCO₃ and 3 ml of water are mixed. A solution of 0.67 g (8.8 mmol) ofthioacetic acid and 1.44 g (10.43 mmol) of K₂ CO₃ in 21 ml of water isadded at 0° C. The mixture is stirred for 15 hours at 20° C. It isacidified with aqueous 6N HCl solution. It is extracted twice withether. The combined ether phases are washed with water, dried overMgSO₄, filtered and concentrated. Yield=67% (recrystallized in ether)m.p. 114° C. IR (nujol): 1670 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 9.55 (s, 1H);8.00 (s, 1H); 7.50 (s, 5H); 4.10 (s, 2H); 2.30 (s, 3H).

C) Preparation of (E)-2-acetylthiomethyl-3-phenylpropenoic acid

2 g of the above (Z) acid dissolved in 30 ml of ethanol are irradiatedfor 16 hours using a Hanovia TQ 150 lamp. After evaporation, a Z/E acidmixture is obtained in a ratio of 6/4. This mixture is taken up in 25 mlof ether and a solution of 0.39 g of cyclohexylamine (0.4 eq.) in 5 mlof ether is added. After stirring for 30 minutes, the mixture isfiltered. The recovered salt is treated with aqueous 3N HCl solution.This is extracted with ether. The organic phase is washed with saturatedaqueous NaCl solution, dried over MgSO₄, filtered and concentrated.Yield=32% m.p. 57° C. ¹ H NMR (CDCl₃ /TMS): 9.60 (s, 1H); 7.30 (s, 5H);7.20 (s, 1H); 3.85 (s, 2H); 2.25 (s, 3H).

D) Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate

0.95 g (4 mmol) of (E)-2-acetylthiomethyl-3-phenylpropenoic aciddissolved in 15 ml of anhydrous THF is placed in a round-bottomed flaskfitted with a calcium chloride guard tube. The flask is cooled toapproximately 0°-5° C. in an ice bath and 1.10 g (4 mmol) of benzylβ-alaninate methanesulphonate and 0.41 g (4 mmol) of triethylamine in 5ml of chloroform, a solution of 0.54 g (4 mmol) of hydroxybenzotriazolemonohydrate in 5 ml of THF and a solution of 0.83 g (4 mmol) ofdicyclohexylcarbodiimide in 5 ml of chloroform are successively addedwith stirring. The mixture is allowed to return to room temperature andis stirred for 6 hours.

The precipitate of dicyclohexylurea (DCU) is filtered off and thefiltrate is evaporated to dryness. The pasty residue is taken up inethyl acetate (12 ml). The DCU which has again precipitated is filteredoff. The organic phase is washed successively with water (1×10 ml), withsaturated aqueous sodium hydrogen carbonate solution (3×10 ml), withwater (1×10 ml) and with saturated aqueous NaCl solution (1×10 ml). Itis dried over MgSO₄, filtered and concentrated. A solid white residue isobtained which is dissolved in the minimum of ether. Afterrecrystallization, 1.25 g of a white solid are obtained. Yield: 78%(recrystallized in ether) m.p. 62° C. IR (nujol): 3220, 1730, 1670,1650, 1620 cm⁻¹ ¹ H NMR (CDCl₃): 7.35 to 7.15 (m, 10H); 6.80 (s, 1H);5.95 (t, 1H, J=5.1 Hz); 4.95 (s, 2H); 3.85 (d, 2H, J=1 Hz); 3.45 (q app,2H, J=6.10 Hz); 2.45 (t, 2H, J=6.0 Hz); 2.30 (s, 3H) ¹³ C NMR (CDCl₃):194.8; 171.7; 168.2; 135.4; 134.9; 134.2; 132.1; 128.5; 128.3; 128.1;66.3; 34.5; 33.4; 33.1; 30.5. Microanalysis: C₂₂ H₂₃ O₄ NS Calc. %C=66.48 H=5.83 N=3.52 Found. % C=66.34 H=5.82 N=3.53

Example 2: Preparation of methylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (E)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step C) is coupled with methyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 60%(after flash chromatography, eluent: ether/petroleum ether 6/4) m.p. 54°C. IR (nujol): 3280, 1720, 1680, 1630, 1610 cm⁻¹ ¹ H NMR: 7.20, (s, 5H);6.80 (s, 1H); 5.90 (broad s, 1H); 3.85 (s, 2H); 3.50 (s, 3H); 3.60 to3.30 (m, 2H); 2.35 (t, 2H, J=5.30 Hz); 2.25 (s, 3H). Microanalysis: C₁₆H₁₉ O₄ NS Calc. % C=59.79 H=5.96 N=4.36 Found. % C=59.67 H=6.28 N=4.47

Example 3: Preparation of ethylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (E)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step C) is coupled with ethyl β-alaninate according to the experimentalprocedure described in Example 1 (step D). Yield=68% (chromatography onsilica, eluent: ether/petroleum ether 6/4) m.p.<50° C. IR (nujol): 3300,1730, 1690, 1640, 1610 cm⁻¹ ¹ H NMR: (CDCl₃ /TMS)=7.20, (s, 5H); 6.75(s, 1H); 6.00 (broad s, 1H); 3.95 (q, 2H, J=6, 7 Hz); 3.85 (s, 2H); 3.40(q app., 2H, J app.=6.2 Hz); 2.30 (t, 2H, J=6.7 Hz); 2.30 (s, 3H); 1.15(t, 3H, J=6.7 Hz). Microanalysis: C₁₇ H₂₁ O₄ NS Calc. % C=60.87 H=6.31N=4.18 Found. % C=60.12 H=6.22 N=3.92

Example 4: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]-β-alanine

To a solution of 2 mmol of the diester obtained in Example 1 (step D)dissolved in a THF/H₂ O (75/25) mixture, are added, at 0° C. and underan argon atmosphere, 8 mmol of LiOH with stirring for 2 hours. The THFis evaporated off and the aqueous phase is washed with ether andacidified with aqueous 3N HCl solution. It is extracted with ether andthe ether extract is washed with saturated aqueous NaCl solution anddried over MgSO₄. It is filtered and evaporated. Yield: 63% (after flashchromatography, eluent: ether) m.p. 80° C. IR (nujol): 3340, 1700, 1630,1610 cm⁻¹. ¹ H NMR (acetone D6): 10.90 to 10.60 (m, 1H); 7.65 (m, 1H);7.45 to 7.35 (m, 5H); 7.20 (s, 1H); 3.75 to 3.50 (m, 4H); 2.60 (t, 2H,J=6.75 Hz); 2.30 (t, 1H, J=7.50 Hz) Microanalysis: C₁₃ H₁₅ O₃ NS Calc. %C=58.85 H=5.70 N=5.28 Found. % C=58.68 H=5.78 N=5.17

Example 5: Preparation of benzylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3-phenylropenyl]-β-alaninate A)Preparation of (Z)-2-benzoylthiomethyl-3-phenylpropenoic acid

The (Z)-2-bromomethyl-3-phenylpropenoic acid described in Example 1(step A) is reacted with thiobenzoic acid according to the experimentalprocedure described in Example 1 (step B). Yield=67% (recrystallized inether) m.p. 160° C. ¹ H NMR CDCl₃ /TMS): 8.15 to 7.80 (m, 3H); 7.70 to7.20 (m, 8H); 4.20 (s, 2H)

B) Preparation of the (Z/E) mixture of2-benzoylthiomethyl-3-phenylpropenoic acid

2 g of the above (Z) acid dissolved in 20 ml of ethanol are irradiatedfor 16 hours using a Hanovia TQ 150 lamp. After evaporation, a Z/E acidmixture (60/40) is obtained. ¹ H NMR (CDCl₃ /TMS): 9.00 (s, 1H); 8.20 to6.8 (m, 11H); 4.25 (S, 1.2H); 4.10 (s, 0.8H).

C) Preparation of benzylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (Z/E)-2-benzoylthiomethyl-3-phenylpropenoic acid described inExample 5 (step B) is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). The (E) isomeris subsequently purified by flash chromatography using the mixtureether/petroleum ether (50/50) as eluent. Yield: 16% m.p. 60° C. IR(nujol): 3320, 1720, 1650, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS)=8.05 to 7.80(m, 2H); 7.60 to 7.00 (m, H), 6.90 (s, 1H), 6.00 (broad t, 1H, J=6.2Hz); 4.90 (s, 2H); 4.05 (s, 2H); 3.4 (q app, 2H, J app.=6.7 Hz); 2.40(t, 2H, J=5.3 Hz). Microanalysis: C₂₇ H₂₅ O₄ NS Calc. % C=70.57 H=5.48N=3.05 Found. % C=70.18 H=5.63 N=2.86

Example 6: Preparation of methylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (Z/E)-2-benzoylthiomethyl-3-phenylpropenoic acid described inExample 5 (step B) is coupled with methyl β-alaninate according to theexperimental procedure described in Example 1 (step D). The (E) isomeris subsequently purified by flash chromatography with the mixtureether/petroleum ether (60/40) as eluent. Yield=16% m.p. 66° C. IR(nujol): 3280, 1730, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃): 8.10 to 7.80 (m,2H); 7.70 to 7.10 (m, 8H); 6.90 (s, 1H); 6.00 (broad t, 1H); 4.05 (s,2H); 3.70 to 3.10 (m, 2H); 3.40 (s, 3H); 2.35 (t, 2H, J=5.4 Hz).Microanalysis: C₂₁ H₂₁ O₄ NS Calc. % C=65.78 H=5.52 N=3.65 Found. %C=65.51 H=5.75 N=3.20

Example 7: Preparation of ethylN-(E)-[1-oxo-2(benzoylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (Z/E)-2-benzoylthiomethyl-3-phenylpropenoic acid described inExample 5 (step B) is coupled with ethyl β-alaninate according to theexperimental procedure described in Example 1 (step D). The (E) isomeris subsequently purified by flash chromatography with the mixtureether/petroleum ether (60/40) as eluent. Yield=13% m.p.<50° C. ¹ H NMR(CDCl₃): 8.10 to 7.80 (m, 2H); 7.70 to 7.10 (m, 8H); 6.90 (s, 1H); 6.00(broad t, 1H); 4.05 (s, 2H); 3.90 (q, 2H, J=7.5 Hz); 3.45 (q app, 2H, Japp=5.3 Hz); 2.35 (t, 2H, J=5.3 Hz); 1.10 (t, 3H, J=7.5 Hz)Microanalysis: C₂₂ H₂₃ O₄ NS Calc. % C=66.48 H=5.83 N=3.52 Found. %C=66.80 H=5.92 N=3.75

Example 8: Preparation of benzylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate A)Preparation of (Z)-2-pivaloylthiomethyl-3-phenylpropenoic acid

The (Z)-2-bromomethyl-3-phenylpropenoic acid described in Example 1(step A) is reacted with thiopivaloic acid according to the experimentalprocedure described in Example 1 (step B). Yield: 93% ¹ H NMR (CDCl₃):9.10 (broad s, 1H); 7.90 (s, 1H); 7.35 (s, 5H); 4.00 (s, 2H); 1.20 (s,9H)

B) Preparation of (E)-2-pivaloylthiomethyl-3-phenylpropenoic acid

The (Z)-2-pivaloylthiomethyl-3-phenylpropenoic acid above is irradiatedaccording to the experimental procedure described in Example 1 (step C).Purification of the (E) isomer is carried out according to theexperimental procedure described in Example 1 (step C). Yield: 24%(relative to the starting (Z) acid) m.p. 88° C. ¹ H NMR (CDCl₃): 8.70(broad s, 1H); 7.45 to 7.10 (m, 6H); 3.85 (s, 2H); 1.20 (s, 9H).

C) Preparation of benzylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (E)-2-pivaloylthiomethyl-3-phenylpropenoic acid above is coupledwith benzyl β-alaninate according to the experimental proceduredescribed in Example 1 (step D). Yield: 63% (after flash chromatography,eluent: ether/petroleum ether 55/45) oil IR: 3320, 1725, 1650, 1620 cm⁻¹¹ H NMR (CDCl₃): 7.30 (s, 5H); 7.20 (s, 5H); 6.75 (s, 1H); 4.90 (s, 2H);3.80 (s, 2H); 3.45 (q app; 2H, J=6.0 Hz); 2.40 (t, 2H, J=6.5 Hz); 1.20(s, 9H). Microanalysis: C₂₅ H₂₉ NO₄ S Calc. % C=68.31 H=6.65 N=3.19Found. % C=68.02 H=6.73 N=3.01

Example 9: Preparation of methylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (E)-2-pivaloylthiomethyl-3-phenylpropenoic acid described in Example8 (step B) is coupled with methyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 69%(after flash chromatography, eluent: ether/petroleum ether 55/45) oilIR: 3300, 1730, 1660, 1630 cm⁻¹ ¹ H NMR (CDCl₃): 7.20 (s, 5H); 6.75 (s,1H); 5.90 (broad t, 1H); 3.80 (s, 2H); 3.50 (s, 3H); 3.60 to 3.25 (m,2H); 2.40 (t, 2H, J=6.5 Hz); 1.20 (s, 9H) Microanalysis: C₁₉ H₂₅ NO₄ SCalc. % C=62.79 H=6.93 N=3.85 Found. % C=63.20 H=7.10 N=3.92

Example 10: Preparation of ethylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (E)-2-pivaloylthiomethyl-3-phenylpropenoic acid described in Example8 (step B) is coupled with ethyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 73%(after flash chromatography, eluent: ether/petroleum ether 55/45)-oilIR: 3300, 1710, 1650, 1620 cm⁻¹ ¹ H NMR (CDCl₃): 7.20 (s, 5H); 6.75 (s,1H); 6.0 (broad t, 1H); 3.95 (q, 2H, J=7.5 Hz); 3.80 (s, 2H); 3.40 (qapp., 2H, J. app=6.40 Hz); 2.35 (t, 2H, J=5.9 Hz); 1.30 to 0.95 (m,12H). Microanalysis: C₂₀ H₂₇ NO₄ S Calc. % C=63.63 H=7.21 N=3.71 Found.% C=63.35 H=7.16 N=3.52

Example 11: Preparation of methyl3-(RS)-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl)]aminobutanoate

The (E)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step C) is coupled with methyl 3-(RS)-aminobutanoate according to theexperimental procedure described in Example 1 (step D). Yield: 70%(recrystallized) m.p. 98° C. IR (nujol): 3300, 1730, 1700, 1620 cm⁻¹ ¹ HNMR (CDCl₃): 7.20 (s, 5H); 6.80 (s, 1H); 5.90 (broad d, 1H); 4.50 to4.10 (m, 1H); 3.85 (s, 2H); 3.50 (s, 3H); 2.30 (s, 5H); 1.00 (d, 3H,J=6.4 Hz). Microanalysis: C₁₇ H₂₁ NO₄ S Calc. % C=60.87 H=6.31 N=4.18Found. % C=60.34 H=6.27 N=3.98

Example 12: Preparation of3-(RS)-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]aminobutanoicacid

Saponification of the above diester is carried out according to theexperimental procedure described in Example 4. Yield: 63% (oil) IR:3280, 1700, 1640, 1600 cm⁻¹ ¹ H NMR (CDCl₃): 10.0 (s, 1H); 7.20 (s, 5H);6.65 (s, 1H); 6.15 (d, 1H, J=9.8 Hz); 4.80 to 4.10 (m, 1H); 3.5 (d, 2H,J=8.4 Hz); 2.40 (d, 2H, J=5.6 Hz): 1.75 (t, 1H, J=8.4 Hz); 1.00 (d, 3H,J=7.0 Hz). Microanalysis: C₁₄ H₁₇ NO₃ S Calc. % C=60.19 H=6.13 N=5.01Found. % C=59.39 H=6.56 N=4.71

Example 13: Preparation of methyl3-(RS)-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]amino-2-methylpropanoate

The (E)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step C) is coupled with methyl 3-(RS)-amino-2-methylpropanoateaccording to the experimental procedure described in Example 1 (step D).Yield: 60% (after flash chromatography), eluent: ether/petroleum ether6/4) ¹ H NMR (CDCl₃): 7.40 to 7.15 (m, 5H); 6.75 (s, 1H); 5.90 (broad t,1H); 3.85 (s, 2H); 3.55 to 3.35 (m, 1H); 3.50 (s, 3H); 3.25 to 3.05 (m,1H); 2.65 to 2.45 (m, 1H); 2.35 (s, 3H); 1.05 (d, 3H, J=7.5 Hz)Microanalysis: C₁₇ H₂₁ NO₄ S Calc. % C=60.87 H=6.31 N=4.18 Found. %C=60.53 H=6.28 N=4.30

Example 14: Preparation of3-(RS)-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]amino-2-methylpropanoicacid

Saponification of the diester described in Example 13 is carried outaccording to the experimental procedure described in Example 4. Yield:60% (after flash chromatography, eluent: ether)-oil ¹ H NMR (CDCl₃):7.40 to 7.10 (m, 6H); 6.85 (broad t, 1H); 6.55 (s, 1H); 3.65 to 3.30 (m,4H); 2.55 to 2.40 (m, 1H); 1.70 (t, 1H, J=7.5 Hz); 1.05 (d, 3H, J=7.5Hz) Microanalysis: C₁₄ H₁₇ NO₃ S Calc. % C=60.19 H=6.13 N=5.01 Found. %C=59.48 H=5.99 N=4.79

Example 15: Preparation of benzyl4-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]aminobutanoate

The (E)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step C) is coupled with benzyl 4-aminobutanoate according to theexperimental procedure described in Example 1 (step D). Yield: 62%(after flash chromatography), eluent: ether/petroleum ether 7/3 ) m.p.86° C. IR (nujol): 3300, 1720, 1685, 1640, 1610 cm⁻¹ ¹ H NMR (CDCl₃):7.45 to 7.15 (m, 10H); 6.80 (s, 1H); 5.55 (broad t, 1H); 5.05 (s, 2H);3.85 (s, 2H); 3.20 (q. app., 2H, J=6.0 Hz); 2.30 (s, 3H); 2.20 (t, 2H,J=7.5 Hz; 1.75 to 1.50 (m, 2H). Microanalysis: C₂₃ H₂₅ NO₄ S Calc. %C=67.13 H=6.12 N=3.40 Found. % C=67.07 H=6.07 N=3.46

Example 16: Preparation of4-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]aminobutanoic acid

Saponification of the diester described in Example 15 is carried outaccording to the experimental procedure described in Example 4. Yield:58% (after flash chromatography, eluent: ether)-oil ¹ H NMR (CDC;₃): 7.4to 7.10 (m, 6H); 6.65 (s, 1H); 5.70 (broad s, 1H); 3.50 (d, 2H, J=7.5Hz); 3.25 (q app., 2H, J=6 Hz); 2.20 (t, 2H, J=7.5 Hz); 1.80 to 1.55 (m,3H). Microanalysis: C₁₄ H₁₇ NO₃ S Calc. % C=60.19 H=6.13 N=5.01 Found. %C=60.60 H=6.32 N=5.31

Example 17: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-methoxyphenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(4-methoxyphenyl)propenoic acid

A mixture of 68 g of p-anisaldehyde (500 mmol), 81 g of propionicanhydride (625 mmol) and 48 g of sodium propionate (500 mmol, dried for5 h at 120° C.) is heated under argon for 30 h at 140° C. This solution,cooled to 80° C., is poured slowly into 400 ml of vigorously stirredsaturated aqueous sodium hydrogen carbonate solution. After extractionwith dichloromethane (twice 300 ml), the organic phases are combined andthen washed with saturated sodium hydrogen carbonate solution (twice 150ml). The aqueous phases are combined and then poured slowly into amixture of 300 ml of 12N hydrochloric acid solution and 100 g of crushedice. The precipitate is collected after filtration, dried in adessicator, triturated in twice 150 ml of petroleum ether, filteredagain and dried in the dessicator. 55.70 g (290 mmol) of a white solidare collected. Yield=58% m.p. 178°-182° C. IR (nujol): 1665 cm⁻¹ ¹ H NMR(CDCl₃ /TMS): 9.20 to 8.90 (broad s, 1H); 7.60 (s, 1H); 7.35 to 6.90)AB, 4H, J=8 Hz); 3.80 (s, 3H); 2.1 (s, 3H) ¹³ C NMR (CDCl₃): 170.40;158.50, 137.60; 130.90; 128.10; 126.30; 113.50; 54.80.

B) Preparation of (Z)-2-bromomethyl-3-(4-methoxyphenyl)propenoic acid

A mixture of 30 g (156 mmol) of the above acid, 27.80 g (156 mmol) ofN-bromosuccinimide (NBS) and a catalytic amount of benzoyl peroxide in650 ml of CHCl₃ is heated at reflux for 6 hours, under irradiation usinga halogen lamp (500W). After cooling to room temperature, the solutionis washed with 1N hydrochloric acid solution (3 times 200 ml), decantedand then dried over magnesium sulphate, It is filtered, concentrated andtriturated twice in 350 ml of an ether/petroleum ether mixture (1/3).The mother-liquors are evaporated and 39.0 g (144 mmol) of a white solidare collected. Yield=92% (after trituration in an ether/petroleum ethermixture 1/2) m.p. 173° C. IR (nujol): 1670 cm⁻¹ ¹ H NMR (CDCl₃): 9.6 to9.3 (broad s, 1H); 7.8 (s, 1H); 7.55 and 6.90 (AB, 4H, J=8 Hz); 4.4 (s,2H); 3.8 (s, 3H)

C) Preparation of (Z)-2-(acetylthiomethyl)-3-(4-methoxyphenyl)propenoicacid

To a solution of 8.40 g (31 mmol) of the above acid dissolved in 100 mlof tetrahydrofuran at 0° C. is added dropwise a solution of 4.0 g (31mmol) of diisopropylethylamine and 2.36 g (31 mmol) of thioacetic acidin 30 ml of tetrahydrofuran. At the end of the addition, the solution isfiltered and the filtrate is concentrated. The residue is taken up with80 ml of dichloromethane and the resulting solution is washed with 60 mlof 1N hydrochloric acid solution. After decantation, the organic phaseis dried over magnesium sulphate, filtered and concentrated. 8.09 g(30.4 mmol) of a pale yellow solid are recovered. Yield: 98% m.p. 155°C. IR (nujol): 1690, 1675, 1615 cm ⁻¹ ¹ H NMR (CDCl₃): 10.50 to 10.00(broad s, 1H); 7.85 (s, 1H); 7.40 and 6.90 (AB, 4H, J=8 Hz); 4.05 (s,2H); 3.80 (s, 3H); 2.35 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-methoxyphenyl)propenyl]-β-alaninate

The (Z)-2-acetylthiomethyl-3-(4-methoxyphenyl)propenoic acid obtained instep C is coupled with benzyl β-alaninate according to the experimentalprocedure described in Example 1 (step D). Yield: 74% (crystallizationin a dichloromethane/petroleum ether mixture) m.p. 96° C. IR (nujol):3280, 1740, 1690, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.65 (s, 1H):7.35 to 7.15 (m, 7H); 6.95 to 6.80 (m, 3H); 5.10 (s, 2H); 4.00 (s, 2H);3.75 (s, 3H); 3.60 (q, 2H, J app.=6 Hz); 2.60 (t, 2H, J=6 Hz); 2.30 (s,3H). ¹³ C NMR (CDCl₃): 196.2; 172.0; 167.1; 159.8; 137.9; 135.6; 130.9;128.5; 128.2; 128.1; 127.2; 114.1; 66.3; 55.2; 35.6; 33.8; 30.2; 26.6.Microanalysis: C₂₃ H₂₅ O₅ NS Calc. % C=64.61 H=5.89 N=3.27 Found. %C=64.20 H=5.73 N=3.48

Example 18: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-methoxylphenyl)propenyl]-β-alaninate

4.93 g (11.5 mmol) of the (Z) diester obtained in Example 17 (step D)are dissolved in 100 ml of methylene chloride which has been passed overbasic alumina. The solution is irradiated for 2.5 h using a Hanovia TQ150 lamp after having added dropwise 1.53 ml of boron trifluorideetherate solution (11.5 mmol). The solution is then washed with 1Nhydrochloric acid solution (twice 80 ml), with saturated sodiumhydrogencarbonate solution (twice 80 ml) and is dried over magnesium sulphate.After filtration and evaporation, 2.56 g of (E) isomer are recoveredafter flash chromatography. Yield=52% (after flash chromatography,eluent: ether/petroleum ether 65/35) m.p.<50° C. IR (nujol): 3290, 1740,1690, 1630 cm ⁻¹ ¹ H NMR (CDCl₃): 7.35 to 7.05 (m, 7H; 6.75 (AB, 2H, J=8Hz); 6.70 (s, 1H); 6.10 (t, 1H, J=5 Hz); 4.95 (s, 2H); 3.80 (s, 3H);3.40 (q app, 2H, J app.=6 Hz); 2.45 (t, 2H, J=6 Hz); 2.25 (s, 3H). ¹³ CNMR (CDCl₃): 194.8, 171.6; 168.6; 159.4; 135.4; 132.0; 131.6; 129.8;128.5; 128.2; 128.0; 127.2; 113.6; 66.2; 55.0; 34.6; 33.6; 33.2; 30.4Microanalysis: C₂₃ H₂₅ O₅ NS Calc. % C=64.61 H=5.89 N=3.27 Found. %C=64.17 H=5.91 N=3.48

Example 19: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-methoxyphenyl)propenyl]-β-alanin

Saponification of the (E) diester obtained in Example 18 is carried outaccording to the experimental procedure described in Example 4. Yield:69% (after flash chromatography, eluent: ether) m.p. 160° C. IR (nujol):3340, 1730, 1620, 1580 cm⁻¹ ¹ H NMR (CDCl₃): 9.10 (broad s, 1H); 7.75and 7.15 (AB, 4H, J=8Hz); 6.60 (s, 1H); 6.15 (broad t, 1H): 3.75 (s,3H); 3.50 to 3.45 (m, 4H); 2.50 (t, 2H, J=5 Hz): 1.70 (t; 1H, J=7 Hz).Microanalysis: C₁₄ H₁₇ O₄ NS Calc. % C=56.92 H=5.80 N=4.74 Found. %C=57.11 H=5.86 N=4.91

Example 20: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenyl]-.beta.-alaninateA) Preparation of (E)-2-methyl-3-(4-trifluoromethylphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 4-trifluoromethylbenzaldehyde.Yield=88% m.p. 170° C. ¹ H NMR (CDCl₃ /TMS): 10.55 to 10.00 (broad s,1H); 7.75 to 7.35 (m, 5H); 2.05 (s, 3H).

B) Preparation of (Z)-2-bromomethyl-3-(4-trifluoromethylphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 91% m.p. 183° C. IR (nujol): 1665 cm⁻¹ ¹ HNMR (CDCl₃ /TMS): 8.20 to 8.00 (broad s, 1H); 7.80 (s, 1H); 7.70 to 7.55(m, 4H); 4.30 (s, 2H).

C) Preparation of(Z)-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step C). Yield=82% m.p. 132° C. IR (nujol): 1690, 1670 cm⁻¹¹ H NMR (CDCl₃ /TMS): 11.20 to 11.00 (broad s, 1H); 7.90 (s, 1H); 7.70and 7.50 (AB, 4H, J=10 Hz); 4.05 (s, 2H); 2.35 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenyl].beta.-alaninate

The (Z)-2-acetylthiomethyl-3-(4-trifluoromethylphenyl)propenoic acidobtained in step C is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield=47%(purification by flash chromatography, eluent: ether/petroleum ether6/4) m.p. 79° C. IR (nujol): 3280, 1740, 1690, 1640, 1620 cm⁻¹ ¹ H NMR(CDCl₃ /TMS): 7.70 (s, 1H); 7.55 (AB, 2H, J=8 Hz); 7.45 to 7.20 (m, 7H);6.95 (t, 1H, J=6 Hz); 5.15 (s, 2H); 3.90 (s, 2H); 3.40 (q app., 2H, Japp.=6 Hz.); 2.65 (t, 2H, J=5 Hz); 2.30 (s, 3H). Microanalysis: C₂₃ H₂₂O₄ NSF₃ Calc. % C=59.35 H=4.76 N=3.01 Found. % C=59.17 H=4.73 N=2.87

Example 21: Preparation of benzyl-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenyl]-.beta.-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) diester obtained in Example 20 (stepD). Yield=53% (purification by flash chromatography, eluent:ether/petroleum ether 57/43) m.p. 76° C. IR (nujol): 3290, 1740, 1690,1630 cm⁻¹ ¹ H NMR (CDCl₃): 7.45 (AB, 2H, J=8 Hz); 7.35 to 7.20 (m, 7H);6.70 (s, 1H); 6.25 (t, 1H, J=5 Hz); 3.80 (s, 2H); 3.40 (q. app., 2H, Japp.=6 Hz); 2.40 (t, 2H, J=6 Hz); 2.30 (s, 3H). Microanalysis: C₂₃ H₂₂O₄ NSF₃ Calc. % C=59.35 H=4.76 N=3.01 Found. % C=59.01 H=4.51 N=3.24

Example 22: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-trifluoromethylphenyl)propenyl]-.beta.-alanine

Saponification of the (E) diester obtained in Example 21 is carried outaccording to the experimental procedure described in Example 4.Yield=22% m.p. 108° C. IR (nujol): 1730, 1620, 1580 cm ⁻¹ ¹ H NMR(CDCl₃): 8.50 (broad s, 1H); 7.50 to 7.25 (m, 4H); 6.65 (s, 1H); 6.50(broad t, 1H); 3.50 to 3.40 (m, 4H); 2.60 (t, 2H, J=6 Hz); 1.80 (t, 1H,J=8 Hz). Microanalysis: C₁₄ H₁₄ O₃ NSF₃ Calc. % C=50.45 H=4.23 N=4.20Found. % C=50.11 H=3.99 N=4.05

Example 23: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(4-phenylphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 4-phenylbenzaldehyde and using 6equivalents of propionic anhydride. Yield: 49% m.p. 178° C. IR (nujol):1670 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 11.50 to 11.20 (broad s, 1H); 7.70 (s,1H); 7.60 to 7.20 (m, 9H); 2.15 (s, 3H).

B) Preparation of (Z)-2-bromomethyl-3-(4-phenylphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 78% m.p. 16° C. ¹ H NMR (CDCl₃ /TMS): 7.85(s, 1H); 7.75 to 7.20 (m, 9H); 7.00 to 6.70 (broad s, 1H); 4.40 (s, 2H).

C) Preparation of (Z)-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step C). Yield: 97% m.p. 158° C. IR (nujol)=1690, 1670 cm⁻¹¹ H NMR (CDC;₃ /TMS): 10.40 to 10.00 (broad s, 1H); 7.95 (s, 1H); 7.75to 7.20 (m, 9H); 4.15 (s, 2H); 2.35 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenyl]-β-alaninate

The (Z)-2-acetylthiomethyl-3-(4-phenylphenyl) propenoic acid obtained instep C is coupled with benzyl β-alaninate according to the experimentalprocedure described in Example 1 (step D). Yield: 56% (purification byflash chromatography, eluent: ether/petroleum ether 6/4) m.p. 79° C. IR(nujol): 3280, 1740, 1690, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.75(s, 1H); 7.65 to 7.20 (m, 14H); 7.05 (t, 1H, J=5 Hz); 5.0 (s, 2H); 4.15(s, 2H); 3.65 (q. app., 2H, J app.=6 Hz); 2.65 (t, 2H, J=6Hz); 2.30 (s,3H). Microanalysis: C₂₈ H₂₇ O₄ NS Calc. % C=71.01 H=5.75 N=2.96 Found. %C=71.08 H=5.60 N=2.46

Example 24: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) diester obtained in Example 23 (stepD). Yield=38% (purification by flash chromatography, eluent:ether/petroleum ether 6/4) m.p. 76° C. IR (nujol): 3280, 1740, 1690,1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.60 to 7.10 (m, 14H); 6.85 (s,1H); 6.20 to 6.00 (m, 1H); 5.10 (s, 2H); 3.75 (s, 2H); 3.40 (q. app.,2H, J. app.=6 Hz); 2.45 (t, 2H, J=6 Hz); 2.30 (s, 3H). Microanalysis:C₂₈ H₂₇ O₄ NS Calc. % C=71.01 H=5.75 N=2.96 Found. % C=71.29 H=5.51N=2.75

Example 25: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-phenylphenyl)propenyl]-β-alanine

Saponification of the (E) diester obtained in Example 24 is carried outaccording to the experimental procedure described in Example 4. Yield:28% m.p. 125° C. IR (nujol): 1730, 1620, 1580 cm⁻¹ ¹ H NMR (CDCl₃ /TMS):9.70 (broad s, 1H); 7.70 to 6.80 (m, 11H); 3.80 to 3.40 (m, 4H); 2.50(t, 2H, J=6 Hz); 1.70 (t, 1H, J=8 Hz). Microanalysis: C₁₉ H₁₉ O₃ NSCalc. % C=66.84 H=5.61 N=4.10 Found. % C=66.45 H=5.29 N=3.79

Example 26: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(4-ethoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 4-ethoxybenzaldehyde. Yield=54% m.p.180° C. IR (nujol): 1670 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.65 (s, 1H); 7.45and 6.85 (AB, 4H, J=8 Hz); 6.65 to 6.35 (broad s, 1H); 4.05 (q, 2H, J=6Hz); 2.05 (s, 3H); 1.30 (t, 3H, J=6 Hz).

B) Preparation of (Z)-2-bromomethyl-3-(4-ethoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 45% m.p. 205° C. IR (nujol): 1665 cm⁻¹ ¹ HNMR (CDCl₃ /TMS): 10.40 to 10.00 (broad s, 1H); 7.80 (s, 1H); 7.50 and6.90 (AB, 4H, J=8 Hz); 4.40 (s, 2H); 4.05 (q, 2H, J=6 Hz); 1.30 (t, 3H,J=6 Hz).

C) Preparation of (Z)-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step C) Yield: 96% m.p. 145° C. IR (nujol): 1690, 1670 cm⁻¹¹ H NMR (CDCl₃ /TMS): 11.00 (s, 1H); 7.85 (s, 1H); 7.40 and 6.95 (AB,4H, J=8 Hz); 4.25 to 3.90 (m, 4H); 2.35 (s, 3H); 1.35 (t, 3H, J=6 Hz).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alaninate

The (Z)-2-acetylthiomethyl-3-(4-ethoxyphenyl) propenoic acid obtained instep C is coupled with benzyl alaninate according to the experimentalprocedure described in Example 1 (step D). Yield=65% (purification byflash chromatography, eluent: ether/petroleum ether 65/35) m.p. 92° C.IR (nujol); 3290, 1740, 1690, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.45(s, 1H); 7.35 to 7.15 (m, 9H); 7.05 to 6.60 (m, 1H); 5.15 (s, 2H); 4.15to 3.85 (m, 4H); 3.75 to 3.45 (q app., 2H, J app.: 6 Hz); 2.65 (t, 2H,J=7 Hz); 2.30 (s, 3H); 1.35 (t, 3H, J=6 Hz). Microanalysis: C₂₄ H₂₇ O₅NS Calc. % C=65.29 H=6.16 N=3.17 Found. % C=65.37 H=6.11 N=3.29

Example 27: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the above (Z) isomer. Yield=42% (after flashchromatography, eluent: ether/petroleum ether 62/38) m.p. 87° C. IR(nujol): 3290, 1740, 1690, 1630 cm⁻¹ ¹ H NMR (CDC₃ /TMS): 7.40 to 7.00(m, 9H); 6.75 (s, 1H); 6.05 (broad t, 1H); 4.95 (s, 2H); 4.10 to 3.75(m, 4H); 3.50 (q app., 2H, J app.=6 Hz); 2.60 to 2.20 (M, 5H); 1.30 (t,3H, J=7 Hz). Microanalysis: C₂₄ H₂₇ O₅ NS Calc. % C=65.29 H=6.16 N=3.17Found. % C=65.37 H=6.34 N=3.22

Example 28: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alanine

Saponification of the (E) diester obtained in Example 27 is carried outaccording to the experimental procedure described in Example 4. Yield:29% m.p. 148° C. IR (nujol): 1730, 1620, 1580 cm⁻¹ ¹ H NMR (CDCl₃): 8.30(broad s, 1H); 7.75 et 7.15 (AB, 4H, J=8 Hz); 6.55 (s, 1H); 6.15 (t, 1H,J=5 Hz); 3.95 (q, 2H, J=6 Hz); 3.50 to 3.45 (m, 4H); 2.50 (t, 2H, J=6Hz); 1.70 (t, 1H, J=8 Hz); 1.35 (t, 3H, J=6 Hz). Microanalysis: C₁₅ H₁₉O₄ NS Calc. % C=58.23 H=6.19 N=4.53 Found. % C=58.51 H=6.12 N=4.81

Example 29: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-propoxyphenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(4-propoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 4-propoxybenzaldehyde. Yield=60% m.p.171° C. IR (nujol): 1665 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 9.00 to 8.30 (broads, 1H); 7.75 (s, 1H); 7.35 and 6.90 (AB, 4H, J=8 Hz); 3.95 (t, 2H, J=7Hz); 2.15 (s, 3H); 1.90 to 1.65 (m, 2H); 1.00 (t, 3H, J=9 Hz).

B) Preparation of (Z)-2-bromomethyl-3-(4-propoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 73% m.p. 189° C. IR (nujol): 1670 cm⁻¹ ¹ HNMR (CDCl₃ /TMS): 11.50 to 11.00 (broad s, 1H); 7.80 (s, 1H); 7.40 and7.00 (AB, 4H, J=8 Hz); 4.40 (s, 2H); 3.90 (t, 2H, J=7 Hz); 1.80 (q.app., 2H, J app.=7 Hz); 1.00 (t, 3H, J=8 Hz).

C) Preparation of (Z) -2-acetylthiomethyl-3-(4-propoxyphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step C). Yield: 98% m.p. 156° C. IR (nujol): 1690, 1675,1615 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 8.60 to 8.30 (broad s, 1H); 7.90 (s,1H); 7.40 and 6.95 (AB, 4H, J=8 Hz); 4.15 (s, 2H); 3.95 (t, 2H, J=7 Hz);2.35 (s, 3H); 1.80 (q, 2H, J app.=7 Hz); 1.00 (t, 3H, J=8 Hz).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-propoxyphenyl)propenyl]-β-alaninate

The (Z)-2-acetylthiomethyl-3-(4-propoxyphenyl)propenoic acid obtained instep C is coupled with benzyl β-alaninate according to the experimentalprocedure described in Example 1 (step D). Yield=66% (purification byflash chromatography, eluent: ether/petroleum ether 65/35)-oil IR: 3280,1740, 1690, 1645, 1620 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.50 (s, 1H); 7.35 to7.15 (m, 7H); 6.95 to 6.75 (m, 3H); 5.15 (s, 2H); 4.00 to 3.50 (m, 6H);2.65 (t, 2H, J=6 Hz); 2.30 (s, 3H); 1.80 (m, 2H, J app.=8 Hz); 1.00 (t,3H, J=8 Hz). Microanalysis: C₂₅ H₂₉ O₅ NS Calc. % C=65.91 H=6.42 N=3.07Found. % C=65.80 H=6.38 N=2.91

Example 30: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-propoxphenyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) isomer. Yield=47% (purification byflash chromatography, eluent: ether/petroleum ether 60/40)-oil IR: 3290,1740, 1690, 1630 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.55 to 6.65 (m, 10H); 6.15to 5.85 (m, 1H); 5.00 (s, 2H); 4.05 to 3.35 (m, 6 H); 2.60 to 2.30 (m,5H); 1.75 (m, 2H, J app.=7 Hz); 0.95 (t, 3H, J=7 Hz). Microanalysis: C₂₅H₂₉ O₅ NS Calc. % C=65.91 H=6.42 N=3.07 Found. % C=65.90 H=6.53 N=3.21

Example 31: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(3,5-difluorophenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 3,5-difluorobenzaidehyde. Yield=82%m.p. 148° C. IR (nujol): 1695, 1620, 1590 cm⁻¹ ¹ H NMR (CDCl₃ /TMS):11.30 (s, 1H); 7.70 (s, 1H); 7.40 to 6.60 (m, 3H); 2.05 (s, 3H).

B) Preparation of (Z)-2-bromomethyl-3-(3,5-difluorophenyl)propenoic acid

The procedure is performed in an analgous manner to that described inExample 17 (step B). Yield: 63% m.p. 151° C. IR (nujol): 1700, 1620,1590 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 8.50 to 8.30 (broad s, 1H); 7.65 (s,1H); 7.45 to 6.75 (m, 3H); 4.25 (s, 2H).

C) Preparation of (Z)-2-acetylthiomethyl-3-(3,5-difluorophenyl)propenoicacid

The procedure is performed in an analgous manner to that described inExample 17 (step C) Yield: 94% m.p. 139° C. IR (nujol): 1700, 1620, 1590cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 10.35 to 10.15 (broad s, 1H); 7.85 (s, 1H);7.05 to 6.95 (m, 3H); 4.05 (s, 2H); 2.30 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alaninate

The (Z) acid obtained in step C above is coupled with benzyl β-alaninateaccording to the experimental procedure described in Example 1 (step D).Yield: 54% (after flash chromatography, eluent: ether/petroleum ether60/40) m.p. 82° C. IR (nujol): 3300, 1750, 1690, 1650 cm⁻¹ ¹ H NMR(CDCl₃ /TMS): 7.40 to 7.05 (m, 6H); 6.95 to 6.70 (m, 4H); 5.15 (s, 2H);3.90 (s, 2H); 3.65 to 3.45 (m, 2H); 2.65 (t, 2H, J=6 Hz); 2.35 (s, 3H).Microanalysis: C₂₂ H₂₁ O₄ NSF₂ Calc. % C=60.96 H=4.88 N=3.23 Found. %C=60.78 H=4.77 N=3.01

Example 32: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the above (Z) isomer. Yield: 61% (after flashchromatography, eluent: ether/petroleum ether 55/45) m.p. 81° C. IR(nujol): 3300, 1750, 1690, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃): 7.35 to 7.25(m, 5H); 6.80 (d, 2H, J app.=10 Hz); 6.65 (s, 1H); 6.60 (d, 1H, Japp.=10 Hz); 6.10 (t, 1H, J=5 Hz); 5.00 (s, 2H); 3.80 (s, 2H); 3.45 (q,2H, J app.=6 Hz); 2.50 (t, 2H, J=6 Hz); 2.30 (s, 3H). Microanalysis: C₂₂H₂₁ O₄ NSF₂ Calc. % C=60.96 H=4.88 N=3.23 Found. % C=60.80 H=4.70 N=3.03

Example 33: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alanine

Saponification of the (E) diester obtained in Example 32 is carried outaccording to the experimental procedure described in Example 4. Yield:27% m.p. 125° C. IR (nujol): 1725, 1630, 1580 cm⁻¹ ¹ H NMR (CDC₃): 9.40(broad s, 1H); 6.80 to 6.65 (m, 3H); 6.55 (s, 1H); 6.15 (broad t, 1H);3.55 to 3.40 (m, 4H); 2.50 (t, 2H, J=5 Hz); 1.70 (t, 1H, J=8 Hz).Microanalysis: C₁₃ H₁₃ O₃ NSF₂ Calc. % C=51.82 H=4.35 N=4.65 Found. %C=52.11 H=4.26 N=4.42

Example 34: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(1-naphphyl)propenyl]-β-alaninate A)Preparation of (E)-2-methyl-3-(1naphthyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 1 -naphthaldehyde. Yield: 65% m.p.155° C. IR (nujol): 1670 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 11.40 to 10.80(broad s, 1H); 8.20 (s, 1H); 8.00 to 7.70 (m, 3H); 7.65 to 7.20 (m, 4H);2.00 (s, 3H).

B) Preparation of (Z)-2-bromomethyl-3-(1-naphthyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 50% m.p. 216° C. IR (nujol): 1670 cm⁻¹ ¹ HNMR (CDCl₃ /TMS): 8.30 (s, 1H); 8.05 to 7.80 (m, 3H); 7.75 to 7.40 (m,4H); 6.30 to 5.80 (broad s, 1H); 4.30 (s, 2H).

C) Preparation of (Z)-2-acetylthiomethyl-3-(1-naphthyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step C). Yield=97% m.p. 175° C. IR (nujol): 1690, 1670 cm⁻¹¹ H NMR (CDCl₃ /TMS): 10.40 to 10.00 (broad s, 1H); 7.95 (s, 1H); 7.75to 7.20 (m, 9H); 4.15 (s, 2H); 2.35 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(1-naphthyl)propenyl]-β-alaninate

The above (Z) acid is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 57%(after flash chromatography, eluent ether/petroleum ether 60/40). m.p.81° C. IR (nujol): 3290, 1740, 1695, 1640, 1620 cm⁻¹ ¹ H NMR (CDCl₃/TMS): 8.20 (s, 1H); 8.00 to 7.70 (m, 3H); 7.65 to 7.00 (m, 10H); 5.20(s, 2H); 3.90 (s, 2H); 3.65 to 3.45 (m, 2H); 2.65 (t, 2H, J=6 Hz); 2.35(s, 3H). Microanalysis: C₂₆ H₂₅ O₄ NS Calc. % C=69.78 H=5.63 N=3.13Found. % C=69.86 H=5.68 N=3.17

Example 35: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(1-naphthyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) isomer. Yield: 51% (after flashchromatography, eluent: ether/petroleum ether 57/43) m.p. 78° C. IR(nujol): 3290, 1740, 1695, 1630 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 7.95 to 7.75(m, 3H) 7.55 to 7.10 (m, 10H); 5.80 to 5.70 (m, 1H); 5.00 (s, 2H); 4.05(s, 2H); 3.65 to 3.45 (m, 2H); 2.65 (t, 2H, J=6 Hz); 2.35 (s, 3H).Microanalysis: C₂₆ H₂₅ O₄ NS Calc. % C=69.78 H=5.63 N=3.13 Found. %C=69.48 H=5.71 N=3.01

Example 36: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(1-naphthyl)propenyl]-β-alanine

Saponification of the (E) diester obtained in Example 35 is carried outaccording to the experimental procedure described in Example 4. Yield:39% m.p. 133° C. IR (nujol): 1735, 1620, 1575 cm⁻¹ ¹ H NMR (CDCl₃ /TMS):8.00 to 7.75 (m, 3H); 7.55 to 7.10 (m, 6H); 6.30 to 6.20 (m, 1H); 3.55to 3.45 (m, 4H); 2.50 (t, 2H, J=6 Hz); 1.70 (t, 1H, J=8 Hz)Microanalysis: C₁₇ H₁₇ O₃ NS Calc. % C=64.74 H=5.43 N=4.44 Found. %C=64.47 H=5.29 N=4.31

Example 37: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate

The (Z)-2-acetylthiomethyl-3-phenylpropenoic acid described in Example 1(step B) is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 78%(after recrystallization in ether) m.p. 80° C. IR: 3380, 1720, 1670,1630, 1600 cm⁻¹ ¹ H NMR (CDCl₃): 7.60 (s, 1H); 7.45 to 7.15 (m, 10H);6.90 (broad t, 1H; 5.15 (s, 2H); 3.95 (s, 2H); 3.65 (q. app., J:6.1 Hz);2.65 (t, 2H, J=6.1 Hz); 2.35 (s, 3H). Microanalysis: C₂₂ H₂₃ NO₄ S Calc.% C=60.48 H=5.83 N=3.52 Found. % C=66.29 H=5.78 N=3.51

Example 38: Preparation ofN-(Z)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]-β-alanine

0.795 g (2.0 mmol) of the compound obtained in the above step, dissolvedin 6 ml of methanol, is placed in a round-bottomed flask. The flask isflushed with argon and the solution is cooled in an ice bath. 5 ml ofaqueous 1N sodium hydroxide solution is added at approximately 5° C. Themixture is stirred for 2 hours at 20° C. The methanol is evaporated offunder vacuum at a temperature below 35° C. The basic aqueous phase iswashed with ether (twice 10 ml). It is subsequently acidified withaqueous 1N HCl solution to pH 1. It is extracted with ether (twice 10ml). The extraction phases are washed once with water, dried over MgSO₄,filtered and concentrated under vacuum. The residue is dried in adessicator over phosphorus pentoxide in order to remove the acetic acid.It is purified by chromatography on silica (eluent: ether) Yield: 81%m.p. 132° C. IR: 3400, 1680, 1620, 1590 cm⁻¹ ¹ H NMR (acetone D6): 10.8(broad s, 1H); 7.65 (broad t, 1H); 7.60 to 7.20 (m, 5H); 7.20 (s, 1H);3.70 to 3.45 (m, 4H); 2.60 (t, 2H, J=6.7 Hz); 2.30 (t, 1H, J=5.9 Hz).Microanalysis: C₁₃ H₁₅ NO₃ S Calc. % C=58.85 H=5.70 N=5.28 Found. %C=58.62 H=5.72 N=5.21

Example 39: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alaninateA) Preparation of (E)-2-methyl-3-(4-phenoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from 4-phenoxybenzaldehyde and using 5equivalents of propionic anhydride. Yield: 68% IR (nujol): 1665 cm⁻¹m.p. 133° C. ¹ H NMR (CDCl₃ /TMS): 11.95 (broad s, 1H); 7.80 (s, 1H);7.60 to 6.85 (m, 9H); 2.15 (s, 3H).

B) Preparation of (Z)-2-bromomethyl-3-(4-phenoxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 76% m.p. 126° C. IR (nujol): 1670 cm⁻¹ ¹ HNMR (CDCl₃ /TMS): 8.60 (broad s, 1H) , 7.75 (s, 1H); 7.55 to 6.85 (m,9H); 4.40 (s, 2H).

C) Preparation of (Z)-2-acetylthiomethyl-3-(4-phenoxyphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step C). Yield: 100% m.p. 118° C. IR (nujol): 1690, 1675,1615 cm⁻¹ ¹ H NMR (CDCl₃ /TMS): 10.30 (s, 1H); 7.80 (s, 1H); 7.55 to6.85 (m, 9H); 3.80 (s, 2H); 2.30 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alaninate

The above (Z) acid is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 66%(after flash chromatography, eluent: ethyl acetate/petroleum ether30/70) m.p. 79° C. IR (nujol): 1730, 1690, 1645 cm⁻¹ ¹ H NMR (CDCl₃):7.50 (s, 1H); 7.40 to 7.00 (m, 14H); 7.00 (t, 1H, J=6 Hz); 5.10 (s, 2H);4.00 (s, 2H); 3.60 (q. app., 2H, J app.=7 Hz); 2.65 (t, 2H, J=6 Hz);2.35 (s, 3H). ¹³ C NMR (CDCl₃): 196.4; 172.4; 167.2; 158.3; 156.4;138.0; 136.0; 131.4; 130.2; 129.7; 129.6; 128.9; 128.6; 128.5; 124.3;119.9; 118.5; 66.7; 35.9; 34.2; 30.6; 26.8. Microanalysis: C₂₈ H₂₇ O₅ NSCalc. % C=68.69 H=5.56 N=2.86 Found. % C=68.76 H=5.68 N=2.97

Example 40: Preparation of benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) isomer of Example 39 (step D).

Yield: 73% (after flash chromatography, eluent: ethyl acetate/petroleumether 25/75). m.p. 88° C. IR (nujol): 1730, 1705, 1625 cm⁻¹ ¹ H NMR(CDCl₃): 7.30 to 6.90 (m, 14H); 6.70 (s, 1H); 6.10 (t, 1H, J=6 Hz); 5.00(s, 2H); 3.80 (s, 2H); 3.50 (q. app., 2H, J app.=6 Hz); 2.50 (t, 2H, J=6Hz); 2.30 (s, 3H). ¹³ C NMR (CDCl₃): 194.9; 171.8; 168.4; 157.4; 156.4;135.4; 133.2; 131.4; 130.0; 129.8; 129.7; 128.6; 128.3; 128.2; 123.6;119.1; 118.3; 66.4; 34.6; 33.6; 33.3; 30.5. Microanalysis: C₂₈ H₂₇ O₅ NSCalc. % C=68.69 H=5.56 N=2.86 Found. % C=68.72 H=5.58 N=2.79

Example 41: Preparation ofN-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alanine

Saponification of the (E) diester obtained in Example 40 is carried outaccording to the experimental procedure described in Example 4. Yield:55% m.p. 108° C. ¹ H NMR (CDCl₃): 9.60 (broad s, H); 7.30 to 6.80 (m,9H); 6.50 (s, 1H); 6.30 (t, 1H, J=6 Hz); 3.50 to 3.40 (m, 4H); 2.50 (t,2H, J=6 Hz); 1.70 (t, 1H, J=8 Hz). IR (nujol): 3290, 1725, 1660, 1650cm⁻¹ Microanalysis: C₁₉ H₁₉ NSO₄ Calc. % C=63.85 H=5.38 N=3.92 Found. %C=63.66 H=5.14 N=3.69

Example 42: Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,4-methylenedioxyphenyl)propenyl]-.beta.-alaninateA) Preparation of (E)-2-methyl-3-(3,4-methylenedioxyphenyl)propenoicacid

The procedure is performed in an analogous manner to that described inExample 17 (step A), starting from piperonal. Yield: 65% m.p. 205° C. ¹H NMR (DMSOd₆): 10.1 (broad s, 1H); 7.50 (s, 1H); 7.00 (s, 1H); 6.95 (s,2H); 6.05 (s, 2H); 2.00 (s, 3H).

B) Preparation of(Z)-2-bromomethyl-3-(3,4-methylenedioxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step B). Yield: 75% m.p. 158° C. ¹ H NMR (CDCl₃ /TMS): 8.60(s, 1H); 7.70 (s, 1H); 7.30 to 6.70 (m, 3H); 6.00 (s, 2H); 4.35 (s, 2H).

C) Preparation of(Z)-2-acetylthiomethyl-3-(3,4-methylenedioxyphenyl)propenoic acid

The procedure is performed in an analogous manner to that described inExample 17 (step. C). Yield: 86% m.p. 142° C. ¹ H NMR (CDCl₃ /TMS): 8.60(s, 1H); 7.80 (s, 1H); 7.10 to 6.70 (m, 3H); 6.00 (s, 2H); 4.05 (s, 2H);2.30 (s, 3H).

D) Preparation of benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,4-methylenedioxyphenyl)propenyl]-.beta.-alaninate

The above (Z) acid is coupled with benzyl β-alaninate according to theexperimental procedure described in Example 1 (step D). Yield: 61%(after flash chromatography, eluent: ether/petroleum ether 50/50). m.p.76° C. IR (nujol); 1730, 1705, 1620 cm⁻¹ ¹ H NMR (CDCl₃): 7.55 (s, 1H),7.35 (s, 5H); 6.90 (broad s, 1H); 6.80 (broad s, 3H); 6.00 (s, 2H); 5.20(s, 2H); 4.00 (s, 2H); 3.65 (q. app., 2H, J. app=7 Hz); 2.70 (t, 2H, J=6Hz); 2.35 (s, 3H). Microanalysis: C₂₃ H₂₃ O₆ NS Calc. % C=62.57 H=5.25N=3.17 Found. % C=62.76 H=5.48 N=3.23

Example 43: Preparation of benzylN-(E)-[1oxo-2-(acetylthiomethyl)-3-(3,4-methylenedioxyphenyl)propenyl]-.beta.-alaninate

The procedure is performed in an analogous manner to that described inExample 18, starting from the (Z) isomer of Example 42 (step D). Yield:73% (after flash chromatography, eluent: ether/petroleum ether 55/45).m.p. 84° C. IR (nujol): 1735, 1695 cm⁻¹ ¹ H NMR (CDCl₃): 7.30 (broad s,5H); 6.95 to 6.80 (m, 4H); 6.20 (m, 1H); 5.90 (s, 2H); 5.10 (s, 2H);3.85 (s, 2H); 3.50 (q. app., 2H, J app.=6 Hz); 2.50 (t, 2H, J=6 Hz);2.30 (s, 3H). Microanalysis: C₂₃ H₂₃ _(O) ₆ NS Calc. % C=62.57 H=5.25N=3.17 Found. % C=62.48 H=5.06 N=3.25

The results of the biological studies, presented below, demonstrate theenkephalinase-inhibitory properties of the compounds of formula (Ia) and(Ib) in accordance with the invention.

The present invention thus also relates to the pharmaceuticalcompositions which contain, as active principle, the compounds offormula (Ia) or (Ib) in accordance with the invention in therapeuticallyeffective amounts.

BIOLOGICAL STUDY

An assay of the enkephalinase-inhibitory activity (J. Pharmac. Exp.Ther., 1987, 243, 666) of the compounds of formulae (Ia) and (Ib) wasperformed.

The results obtained are presented in the table which follows:

    __________________________________________________________________________    IN VITRO BIOLOGICAL RESULTS ON ENKEPHALINASE                                   ##STR25##                                                                                                (E) Isomer (Z) Isomer                             R.sub.1        AA           IC.sub.50 (nM)                                                                           IC.sub.50 (nM)                         __________________________________________________________________________    Ph                                                                                            ##STR26##   Ex 4                                                                              2.8    Ex 38                                                                             171.0                                              ##STR27##   Ex 12                                                                             7.0                                                           ##STR28##   Ex 14                                                                             10.0                                                          ##STR29##   Ex 16                                                                             6.4                                            ##STR30##                                                                                    ##STR31##   Ex 19                                                                             2.7    Ex 17*                                                                            97.0                                ##STR32##                                                                                    ##STR33##   Ex 22                                                                             12.1                                           ##STR34##                                                                                    ##STR35##   Ex 25                                                                             7.5    Ex 23*                                                                            65.0                                ##STR36##                                                                                    ##STR37##   Ex 28                                                                             11.8   Ex 26*                                                                            0% at 0.3 μM                     ##STR38##                                                                                    ##STR39##   Ex 30*                                                                            0% at 0.3 μM                                ##STR40##                                                                                    ##STR41##   Ex 33                                                                             5.9    Ex 31*                                                                            16% at 0.3 μM                    ##STR42##                                                                                    ##STR43##   Ex 41                                                                             5.8                                            ##STR44##                                                                                    ##STR45##   Ex 43                                                                             19     Ex 42*                                                                            0% at 0.3 μM                    __________________________________________________________________________     *by enzymatic deprotection of the diester                                

These results illustrate the advantageousenkephalinase-inhibitory-properties of the compounds according to theinvention, which make themuseful in human and veterinary medicine.

The pharmaceutical compositions which contain the compounds of formula(Ia) or (Ib) according to the invention are useful in the indicationsresulting from the central properties, in particular as painkillers, andthe peripheral properties, in particular as anti-diarrhoea agents, ofthe enkephalinase inhibitors, as well as in the indications of ANFprotectors, in particular arterial hypertension and cardiacinsufficiency.

The pharmaceutical compositions which contain, as active principle, thecompounds of formula (Ia) or (Ib) in accordance with the invention maybe administered to man via the oral, parenteral or rectal route.

These pharmaceutical compositions may be in solid or liquid form and maybe provided in the pharmaceutical forms commonly used in human medicine,such as, for example, in the form of simple or coated tablets, gelatincapsules, suppositories or injectable preparations.

The pharmaceutical compositions in accordance with the invention may beadministered in unit doses, preferably of 20 to 200 mg of activeprinciple.

We claim:
 1. Amino acid derivatives, characterized in that theycorrespond to the general formulae ##STR46## in which R₁ represents ahydrogen atom, a phenyl group which is optionally mono- orpolysubstituted with a halogen atom, a trifluoromethyl group, a nitrogroup, a cyano group or an amino group, a lower alkyl group or a lowerphenylalkylene group; the group ##STR47## where R'₁ represents ahydrogen atom, a lower alkyl group, a phenyl group or a lowerphenylalkylene group; a group ##STR48## where A, B and n₃ have themeanings given below

    ______________________________________                                        A               B      n.sub.3                                                ______________________________________                                        O               O      1                                                      O               CH.sub.2                                                                             1                                                      CH.sub.2        CH.sub.2                                                                             1                                                      O               O      2                                                      CH.sub.2        CH.sub.2                                                                             2                                                      O               CH.sub.2                                                                             2                                                      ______________________________________                                    

a biphenyl group, alpha and beta naphthyl, n₁ varies from 0 to 10 n₂varies from 1 to 10 R₂ represents a hydrogen atom; a lower alkyl group;a lower hydroxyalkylene group; a phenyl group; a lower phenylalkylenegroup; a lower hydroxyphenylalkylene group; a lower aminoalkylene group;a lower guanidinoalkylene group; a lower mercaptoalkylene group; a lowerthioalkylene lower alkyl group; a lower imidazolylalkylene group; alower indolylalkylene group; a lower carbamylalkylene group; a lowercarboxyalkylene group; R₃ also represents a hydrogen atom or one of thegroups mentioned above for the definition of R₂ ; R₄ represents ahydrogen atom, a linear or branched aliphatic acyl radical, an aromaticacyl radical which is optionally mono- or polysubstituted, or a linearor branched acyl radical containing one or more oxygen atoms; R₅represents a hydrogen atom; a linear or branched lower alkyl group; aphenyl group or a lower phenylalkylene group, the two last-mentionedgroups being optionally mono- or polysubstituted on the phenyl ring; alinear or branched substituent containing one or more oxygen atoms. 2.Amino acid derivatives according to claim 1, characterized in that R₁represents a phenyl group which is optionally mono- or polysubstitutedwith a halogen atom or with the trifluoromethyl group; the group##STR49## where R'₁ represents a lower alkyl group, a phenyl group; thegroup ##STR50## where A and B represent oxygen and n₃ is equal to 1 or2; a biphenyl group, alpha and beta naphthyl;R₂ and R₃ represent ahydrogen atom or a lower alkyl radical; R₄ represents a hydrogen atom, alinear or branched aliphatic acyl radical or an aromatic acyl radical;R₅ represents a hydrogen atom, a lower alkyl radical or a phenylradical; n₁ is equal to 0 or 1; n₂ is equal to 1 or
 2. 3. Amino acidderivatives according to claim 1, characterized in that they are chosenfrom:benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate, methylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate, ethylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]-β-alanine, benzylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3-phenylpropenyl]-β-alaninate, methylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3-phenylpropenyl]-β-alaninate, ethylN-(E)-[1-oxo-2-(benzoylthiomethyl)-3phenylpropenyl]-β-alaninate, benzylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate,methylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate, ethylN-(E)-[1-oxo-2-(pivaloylthiomethyl)-3-phenylpropenyl]-β-alaninate,methyl3-(RS)-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]aminobutanoate,3-(RS)-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]aminobutanoicacid, methyl3-(RS)-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]amino-2-methylpropanoate,3-(RS)-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]amino-2-methylpropanoicacid, benzyl4-N-(E)-[1-oxo-2-(acetylthiomethyl)-3-phenylpropenyl]aminobutanoate,4-N-(E)-[1-oxo-2-(mercaptomethyl)-3-phenylpropenyl]aminobutanoic acid,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-methoxyphenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-methoxyphenyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-methoxyphenyl)-propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenyl]-.beta.-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-trifluoromethylphenyl)propenyl]-.beta.-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-trifluoromethylphenyl)propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenylphenyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-phenylphenyl)propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-ethoxyphenyl)propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-propoxyphenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-propoxyphenyl)propenyl]-β-alaninate,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(3,5-difluorophenyl)propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(1naphthyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(1naphthyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(1-naphthyl)propenyl]-β-alanine,benzyl N-(Z)-[1-oxo-2-(acetylthiomethyl)-3(phenyl)propenyl]-β-alaninate,N-(Z)-[1-oxo-2-(mercaptomethyl)-3-(phenyl)propenyl]-β-alanine, benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(4-phenoxyphenyl)propenyl]-β-alaninate,N-(E)-[1-oxo-2-(mercaptomethyl)-3-(4-phenoxyphenyl)-propenyl]-β-alanine,benzylN-(Z)-[1-oxo-2-(acetylthiomethyl)-3-(3,4-methylenedioxyphenyl)propenyl]-.beta.-alaninate,benzylN-(E)-[1-oxo-2-(acetylthiomethyl)-3-(3,4-methylenedioxyphenyl)propenyl]-.beta.-alaninate.4. Medicament exhibiting an enkephalinase-inhibitory activity,characterized in that it contains, as active principle, a compoundaccording to claim
 1. 5. Medicament according to claim 4, characterizedin that it is provided in the form of unit doses containing from 20 to200 mg of active principle.